The long-term goal of this project remains the elucidation of the biological functions of glycosyl moieties on glycoconjugates important to development and immunity. During studies on lymphocytes, a new type of protein glycosylation (O-GlcNAc) was discovered. O-GlcNAc occurs in all eukaryotes from yeast to man on a myriad of nuclear and cytoplasmic proteins, including RNA polymerase II and its transcription factors, numerous chromatin-, nuclear pore-, cytoskeletal-, viral-, nuclear oncogene-, and heat shock-proteins. O-GlcNAc appears to be ubiquitous, abundant, and as dynamic as phosphorylation. Elucidation of O-GlcNAc's functions will be the focus of this project's next phase. Our plan is to: 1) Complete the characterization, clone the genes, and understand the regulation, of the enzymes that attach or remove O-GlcNAc. Complexity of gene families, regulation of the enzymes at the genomic, mRNA, and protein levels will be determined. Recombinant enzymes will be used to develop inhibitors, and to study the enzymes' structures. 2) Several approaches will evaluate the general significance of O-GlcNAc: A. CHO-mutant - Cytoplasmic constructs of Gal transferase cDNA will be transfected into gal-deficient CHO cells (LDL-d), and the deleterious effects of exogenous galactose on viability, growth, nuclear transport, transcription, and other properties will be evaluated. Preliminary data suggests that similar cytoplasmic constructs are lethal in wild-type cells. B. Yeast - O-GlcNAc transferase cDNA from yeast will be cloned. Classical gene knock-out experiments will be performed. Effects of glucosamine deprivation on O-GlcNAc will be studied in a yeast Ts auxotrophic for GlcN. C. Inhibitors - Inhibitors of O-GlcNAc transferase, such as polyoxins, and inhibitors of O-GlcNAcase, such as NAc-deoxynorjirimycin, will be studied. D. Anti-Sense - Overexpression of anti-sense cDNA for O-GlcNAc transferase and O-GlcNAcase will also be attempted to specifically effect O-GlcNAc metabolism. 3) Determine the relationship between O-GlcNAc and Ser/Thr phosphorylation: Initially, we will co-examine synthetic peptide substrates with pure O-GlcNAc transferase and purified kinases. Co-localization of sites on endogenous proteins and effects of phosphatase/kinase inhibitors on O-GlcNAc and vice versa will be determined. 4) Continue to Identify O-GlcNAc Attachment Sites for Site-Directed Mutagenesis: Initially, we will focus on RNA polymerase II, serum response and HNF-1 transcription factors, and Neurofilaments. 5) Dynamics of O-GlcNAc will be further explored in cellular activation, cell cycle, and developmental systems. Overall, these experiments will elucidate the functions of O-GlcNAc, a ubiquitous, abundant, and dynamic post-translational modification, which may have substantial impact on nearly all aspects of eukaryotic biology, including development and intracellular processes.